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Unusual Outbreak of Fatal Clostridiosis in a Group of Captive Brown Pelicans (Pelecanus occidentalis).

Abstract: Fatal clostridial infections and clostridial toxicoses are common in birds. Most fatalities are associated with toxin production and progress rapidly, often within 24 hours of infection. We describe an unusual and protracted course of disease in 6 captive brown pelicans (Pelecanus occidentalis), which was believed to result from toxicosis by toxovar A produced by a mixed infection with Clostridium sordellii and Clostridium perfringens. Although the first death in the group occurred 3 days after signs of illness were documented, the remaining birds died over a 38-day period despite aggressive antibiotic and supportive therapy. Although the birds presented with classic signs of botulism, Clostridium botulinum was not identified in any tissues or environmental samples. Postmortem findings in all pelicans included extensive subacute myonecrosis, enteritis, and nonsuppurative hepatitis. Alpha-toxins and sordellilysin genes from C perfringens and C sordelli isolates, respectively, were detected via polymerase chain reaction. The source of the pathogenic bacteria was sediment within a water basin inside the affected birds' enclosure.

Key words: Clostridium sordellii, Clostridium perfringens, avian clostridiosis, myonecrosis, enteritis, cytotoxins, avian, brown pelican, Pelecanus occidentalis

Clinical Report

A captive-bred flock of 4 male and 2 female brown pelicans (Pelicanus occidentalis), ages 1 to 3 years and weighing 2.9 to 4.4 kg, arrived at a zoological facility in Europe in February 2014. The pelicans were fed 3 times per day with frozen thawed fish, usually smelt and sometimes herring or mackerel. The birds were housed in an enclosure measuring 10 X 11 m, which was fully netted. The outer area was connected to a greenhouse measuring 6 X 10 m, which was used as an indoor enclosure. The outdoor area had a pebbled substrate, and the greenhouse floor was covered with wood chips. Two ponds within the enclosure, a larger one outdoors and a smaller one indoors, contained 57 and 15 [m.sup.3] of water, respectively.

On day 1, 6 months after the bird's arrival, male pelican A became weak and had progressive difficulties standing and walking. The bird was separated for examination. A venous blood sample was collected and submitted for hematologic evaluation, results of which showed hemoconcentration (suspected to be the result of dehydration), anemia, and leukopenia with a slight monocytosis and lymphocytopenia (Table 1). The bird was treated empirically with marbofloxacin (10 mg/kg IM q24h), terbinafine (15 mg/kg PO ql2h; Lamisil; Novartis Pharma GmbH, Nuremberg, Germany), meloxicam (0.5 mg/kg PO ql2h), and fluid therapy. However, on day 2, the bird became anorexic and was found dead on the morning of day 3.

Postmortem examination revealed a pelican in good nutritional state weighing 3.0 kg. The proventriculus and ventriculus were empty, and the large intestine was minimally filled with soft greenish material. Multiple hematomas were observed on the breast muscles as well as superficial leg muscles. Histopathologic examination of the latter lesions revealed myofibril necrosis with Zenker's degeneration and a heterophilic, lymphocytic, plasmacytic inflammatory reaction. Connective tissue hyperplasia also was noted. The small and large intestines demonstrated a catarrhal inflammatory reaction. Mild, nonsuppurative hepatitis was present and was characterized by perivascular inflammation. Samples from the liver, heart, kidney, lungs, and small intestine were submitted for bacterial and fungal culture. All tissues were negative for fungal organisms, Salmonella species, Yersinia species, and Listeria monocytogenes. Cultures of submitted colorectal swabs were negative for Campylobacter species but positive for Clostridium species.

On day 4, before final culture results were available from pelican A, female pelican B became ill, exhibiting pharyngeal paralysis, weakness, and uncoordinated movements when attempting to catch and eat fish. The bird's clinical appearance suggested botulism. A blood sample was submitted for hematologic and serum biochemical analysis, and treatment with erythromycin was instituted (50 mg/kg PO ql2h). Erythromycin was chosen because, as a bacteriostatic antibiotic, its activity does not result in overwhelming bacterial destruction and toxin release. Clinical pathology results revealed leukocytosis with slight monocytopenia, and markedly elevated concentrations of liver and muscle enzymes (Table 1).

On day 5, mild paralysis and uncoordinated movements were observed in pelicans C and D. Subsequently, each pelican was treated with erythromycin (similar dosage as pelican B), lactulose (1 mL PO q24h; Lactulose-Saar Sirup; MIP Pharma GmbH, Blieskastel, Germany), parenteral fluid therapy, and pre- and probiotics administered in fish to displace intestinal Clostridia (2.5 g q24h; PT-12 Lactobacillus salivarius; re-scha Biol Pharmaceuticals, Biiren, Germany; and 2 g q24h; Bird Bene-Bac; Albrecht GmbH, Aulendorf, Germany, respectively).

Despite treatment, the condition of pelican B began to worsen on day 7. Pelicans E and F also became weak, anorexic, and dysphagic. At the same time, results from the anaerobic culture of intestinal tissues from pelican A showed growth of Clostridium baratii. While C baratii has the potential to produce botulinum-like neurotoxins, (1,2) neither the botulinum toxin itself nor its encoding genes were identified.

Cloacal swabs from all birds were submitted for anaerobic culture immediately after antibiotic treatment was initiated and then again 5 days after treatment (ie, on day 12 of the course of the disease). Culture of cloacal swabs retrieved on day 12 from pelicans D to F demonstrated high concentrations of Clostridium perfringens. Antibiotic therapy then was switched to benzylpenicillin (20 mg/kg PO ql2h; Aviapen; Medistar Arzneimittelvertrieb GmbH, Bernburg, Germany), which is bactericidal.

Pelicans B and C died on days 13 and 15, respectively. Liver and intestine samples from each bird were submitted for culture and to test for presence of the botulinum neurotoxin genes (BoNT) A and F as well as the nontoxic, nonhemagglutinin (NTNH) used as a surrogate marker for BoNT-producing clostridia. (2,3) All results were negative.

Despite intensive care and hospitalization, pelicans D to F died on days 33, 35, and 38, respectively. All birds were necropsied and tissues were analyzed as for pelican A. In addition, samples of breast muscle were submitted for anaerobic culture. Throughout the disease course, each bird lost 0.6 to 0.9 kg of body weight. All birds had necrotic myositis. Pelicans B to E also had ulcerative diphtheroid inflammation of the colon and rectum.

Postmortem colorectal swabs collected from pelicans B to F and submitted for culture grew large numbers of Clostridium sordellii. In addition. C perfringens toxovar A, positive for C perfringens alpha-toxin gene (cpa), was detected by polymerase chain reaction (PCR) in breast muscle and colorectal tissue from 3 of the 5 birds sampled postmortem. (4)

Environmental samples were obtained from feeder fish as well as the pool water, soil around the pools, and mud from the pool bottom. Low concentrations of C baratii, Clostridium botulinum Group II (nontoxic), and C bifermentans were found in the pool water. Clostridium perfringens was found in high concentrations and C sordellii in low concentrations in mud from the pool bottom. Samples of feeder fish and the enclosure substrate were negative for Clostridia species during anaerobic enrichment culture. By using the primers described by Voth et al, (5) PCR detected the gene encoding the hemolysin, sordellilysin (sdl), in the C sordellii isolate. However, the tested strain was PCR-negative for the lethal toxin gene TcsL. (6)

Discussion

We described an unusual presentation of clostridiosis in a flock of captive brown pelicans, which was characterized by a prolonged course of disease unresponsive to treatment. Although botulism has been a cause of severe mortality in wild pelicans, (7) the botulism-like signs observed in this flock most likely were caused by non-C botulinum clostridial organisms. Very few cases of botulism caused by clostridia other than C botulinum have been described, and all known cases have occurred in humans. (8)

Clostridia are anaerobic, gram-positive, sporeforming bacteria that occur in soil, water, and the mud at lake bottoms, and are commensal organisms in the intestines of animals. Thick-walled, resistant clostridial spores can persist in the environment for years. Clostridia may produce potent bacterial exotoxins, usually causing rapid death. Botulinum neurotoxins (BoNT) produced by C botulinum, particularly toxins C and E, are best known for causing flaccid paralysis in waterfowl. (9,10) However, not all C botulinum produce neurotoxins, as demonstrated by the strain identified from the pool in this report. C baratii and Clostridium butyricum also can produce BoNT; however, in this report, only nontoxinproducing C baratii were identified. Nevertheless, botulism cannot entirely be ruled out as the cause of death of these pelicans, especially in pelican A, because BoNT genes used in diagnosis often are found on a mobile genetic element, such as a plasmid or a phage, which may be lost. (9)

Clostridium occasionally has been associated with gangrenous dermatitis in turkeys" and only recently reported in a case of ulcerative enteritis in quail. (12) While most C sordellii strains solely produce lethal toxin (TcsL), rare C sordellii strains produce hemorrhagic toxin (TcsH) and TcsL. (13) None of these virulence factors was detected in the birds reported in this study. However, genes of the hemolysin sordellilysin (sdl) were identified. Sordellilysin is known to lyse most cell types containing cholesterol-rich membranes, and was shown to demonstrate a 10-fold higher lethal dose ([LD.sub.50]) than TcsL in mice. (5) Hemotoxins may disrupt blood clotting and cause generalized tissue damage, and they are associated with myonecrosis and gangrene in humans. (14) The role of sordellilysin in diseases is not completely clear, (5) but this toxin may have resulted in the prolonged clinical picture observed in this report. Further, TcsL and TcsH are encoded in plasmids, and C sordellii is known to lose the associated plasmid rapidly in vitro. (5,6)

Clostridium sordellii is described rarely as causing disease in waterfowl. In one case involving C sordellii, an Australian pelican (Pelicanus conspicillatus) was found dead among other waterfowl in a pond, demonstrating extensive subcutaneous, pericardial, epicardial, and endocardial hemorrhage as well as hemorrhagic necrotizing enteritis. (15) In that bird, because bacteria were not found outside the intestines, exotoxin involvement was presumed likely. A similar scenario may have taken place in the flock of pelicans described in this report. Although C sordellii is found commonly in the soil and, with low incidence, in the intestines of healthy animals and humans, (14) the almost pure culture of C sordellii from the colorectum of one of the birds described here suggests its role in the disease outbreak.

The necrotic enteritis described in this case could likely be traced back to C perfringens in addition to C sordellii, both of which have cytotoxic activity. C perfringens enteritis apparently is rare in pelicans, (9) although it is very common in poultry and other land birds. (16) Hematoma formation and myonecrosis, as noted in the leg and breast muscles of the pelicans reported here, have been described as common findings in association with C sordellii infections in humans (14) and in a single case reported in a brown bear (Ursus arctos). (17)

Although the main signs of myonecrosis, enteritis, and neurologic abnormalities could be explained by a mixed clostridial infection, it is intriguing that some pelicans remained alive for an extended period. However, eventually all succumbed to the disease. Clostridial infections usually are characterized by a rapid course of clinical disease. The immediate antimicrobial treatment and additional intensive care administered to the birds may have resulted in rapid reduction of the bacteria, but lethal toxin-induced tissue damage already was present. Contaminated pond sediment could have resulted in reinfection, which subsequently killed the remaining birds.

Treatment requires removal of toxigenic Clostridium species from the environment and elimination of the bacteria from the host organism. Bactericidal antimicrobial agents, which can lead to acute and massive bacterial destruction with an overwhelming release of toxins, typically are avoided. Bactericidal and bacteriostatic antibiotics each were administered to these birds with no success, likely because clostridial toxins still were present. Antibiotics that suppress toxin synthesis, such as clindamycin, sometimes are used to treat necrotizing infections due to the possible presence of other toxin-producing, gram-positive organisms. (18) Such antibiotics potentially could have been effective in this case. Our therapy may have failed due to constant recontamination and the inability to eliminate the effects of clostridial cytotoxins. Administration of antitoxins is another potential treatment option, but no commercially available antitoxin currently exists for C sordellii. (14) Vaccination against specific clostridial organisms, which is done commonly for domestic hoofstock, is untested in birds. However, immunization would be worth considering if animals must remain in a contaminated environment.

Certain fish species, such as tilapia, can be an important source of C botulinum and Type C botulism in wild brown and white pelicans (.Pelicanus erythrorhynchos) in North America, causing large die-offs. (7) In the case reported here, 5 different Clostridium species were detected in the water or sediment of the pool bottom but not in any feeder fish. In addition, C botulinum was not found in any sample collected from the pelicans. The finding of C perfringens and C sordellii in cloacal and colorectal swabs collected from the pelicans suggests that fecal contamination of the water basin from the pelicans themselves was the likely source of the contaminated pool sediment.

Environmental clean-up is particularly challenging in cases of clostridial infections. Chlorine was used to disinfect the water, because no filter system or regular water exchange was available. However, clostridial spores are resistant to treatment with chlorine, (20) and the pelicans likely came in contact with the spores while diving for fish thrown into the pond at feeding time. Filtration and regular water exchanges would have been preferable. Decontamination of spores from soil often is an insurmountable problem. Therefore, using concrete pools and routinely removing sludge and other organic debris may be a better option for waterfowl.

Acknowledgments: We thank Liliane and Henk Hiddingh and the animal care team as well as Dr Ellen Wiedner for her valuable scientific input.

References

(1.) Barash JR. Tang TWH. Arnon SS. First case of infant botulism caused by Clostridium baratii type F in California. J Clin Microbiol. 2005:43(8):4280-4282.

(2.) Kirchner S, Kramer KM, Schulze M, et al. Pentaplexed quantitative real-time PCR assay for the simultaneous detection and quantification of botulinum neurotoxin-producing Clostridia in food and clinical samples. Appl Environ Microbiol. 2010; 76(13):4387-4395.

(3.) Raphael BH, Andreadis JD. Real-time PCR detection of the nontoxic nonhemagglutinin gene as a rapid screening method for bacterial isolates harboring the botulinum neurotoxin (A-G) gene complex. J Microbiol Methods. 2007;71 (3):343-346.

(4.) Baums CG, Schotte U, Amtsberg G, Goethe R. Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates. Vet Microbiol. 2004; 100(1-2): 11-16.

(5.) Voth DE. Martinez OV. Ballard JD. Variations in lethal toxin and cholesterol-dependent cytolysin production correspond to differences in cytotoxicity among strains of Clostridium sordellii. FEMS Microbiol Lett. 2006;259(2):295-302.

(6.) Couchman EC, Browne HP, Dunn, M, et al. Clostridium sordellii genome analysis reveals plasmid localized toxin genes encoded within pathogenicity loci. BMC Genomics. 2015; 16(1):392.

(7.) Rocke TE, Nol P, Pelizza C. Sturm KK. Type C botulism in pelicans and other fish-eating birds at the Salton Sea. Studies Avian Biol. 2004;27:136-140.

(8.) Arnon SS. Infant botulism. In: Feigin RD, Cherry JD, Demmler G, Kaplan SL, eds. Textbook of Pediatric Infectious Diseases, 5th ed. Philadelphia, PA: WB Saunders; 2004:1758-1766.

(9.) Redrobe S. Pelecaniformes (pelicans, tropicbirds, cormorants, frigatebirds, anhingas, gannets). In: Miller RE, Fowler ME, eds. Zoo and Wild Animal Medicine. 8th ed. St Louis, MO: Elsevier, 2015: Saunders; 2014:96-99.

(10.) Smith GR. Botulism in waterfowl. Wildfowl. 1976; 27(27): 129-138.

(11.) Dorner MB, Schulz KM, Kull S. Dorner BG. Complexity of botulinum neurotoxins: challenges for detection technology. Curr Top Microbiol Immunol. 2013;364:219-255.

(12.) Clark S, Porter R. McComb B. et al. Clostridial dermatitis and cellulitis: an emerging disease of turkeys. Avian Dis. 2010;54(2):788-794.

(13.) Crespo R, Franca M, Shivaprasad HL. Ulcerative enteritis-like disease associated with Clostridium sordellii in quail. Avian Dis. 2013;57(3):698-702.

(14.) Genth H, Pauillac S, Schelle I, et al. Haemorrhagic toxin and lethal toxin from Clostridium sordellii strain vpi9048: molecular characterization and comparative analysis of substrate specificity of the large clostridial glucosylating toxins. Cell Microbiol. 2014:16(1 0:1706-1721.

(15.) Aldape MJ, Bryant AE, Stevens DL. Clostridium sordellii infection: epidemiology, clinical findings, and current perspectives on diagnosis and treatment. Clin Infect Dis. 2006:43(11): 1436-1446.

(16.) World Organisation for Animal Health. Other 'non listed' diseases of particular interest and unusual mortalities in wildlife. 2009. Available at: http:// www.agriculture.gov.au/SiteCollectionDocuments/ animal-plant/emergency/wildlifeexoticdisease program/08-09/oie-wildlife-diseases-dec05.pdf Accessed August 29, 2017.

(17.) McOrist S, Reece RL. Clostridial enteritis in freeliving lorikeets (Trichoglossus spp.). Avian Pathol. 1992;21 (3):503--507.

(18.) Balseiro A, Oleaga A, Polledo L, et al. Clostridium sordellii in a brown bear (Ursus arctos) from Spain. J Wildl Dis. 2013:49(4): 1047-1051.

(19.) Stevens DL, Maier KA, Laine BM, Mitten JE. Comparison of clindamycin, rifampin, tetracycline, metronidazole, and penicillin for efficacy in prevention of experimental gas gangrene due to Clostridium perfringens. J Infect Dis. 1987; 155(2):220 228.

(20.) Venczel LV, Arrowood M, Hurd M, Sobsey MD. Inactivation of Cryptosporidium parvum oocysts and Clostridium perfringens spores by a mixed-oxidant disinfectant and by free chlorine. Appl Environ Microbiol. 1997:63(4): 1598-1601.

Imke Lueders, DVM, PhD, Carsten Ludwig, DVM, Johanna Kasberg, DVM, Christoph Georg Baums, Prof Dr, Kerstin Klimke, Martin B. Dorner, Prof Dr, Dana Strose, DVM, and Volker Schmidt, DVM, PhD, ECZM (Avian, Herpetology)

From the Westfalischer Zoologischer Garten Munster GmbH, Sentruper Str. 315, 48161 Munster, Germany (Lueders, Ludwig, Kasberg); University of Veterinary Medicine Hannover, Unit for Reproductive Medicine--Clinic for Horses, Biinteweg 15, 30559 Hannover, Germany (Leuders, Kasberg); Institute for Bacteriology and Mycology, Centre for Infectious Diseases, University of Leipzig, An den Tierkliniken 29, 04103 Leipzig, Germany (Baums. Klimke); Consultant Laboratory for Clostridium botulinum, Robert Koch Institute, Seestrassse 10, 13353 Berlin. Germany (Dorner); Tierarztpraxis Dana Strose im Tiergesundheitszentrum Warendorf, Freckenhorster Strae 61, 48231 Warendorf, Germany (Strose); and the Clinic for Birds and Reptiles, University of Leipzig, An den Tierkliniken 17, 04103 Leipzig, Germany (Schmidt).
Table 1. H ematologic and serum biochemical
parameters of 2 pelicans with clostridiosis compared to
reference values. (8)

                                                     Mean reference
Parameter                    Pelican A   Pelican B     values (8)

WBC, cells/[micro]L          8000        16 500      11 300
Lymphocytes, /[micro]L        960          3465        3300
Monocytes, /[micro]L          800           330         639
Heterophils, /[micro]L       6080        12 705        6250
RBC, X [10.sup.9]/[micro]L      2.4           2.7         2.8
Hemoglobin, g/dL               15.3          15.1        15.6
Hematocrit, %                  49            38          46
AST, U/L                      --         2850         301
CK. U/L                       --       16 361        1138
LDH, U/L                      --         8800         893

AST, aspartate aminotransferase: CK, creatine kinase; LDH,
lacatae dehydrogenase.
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Title Annotation:Clinical Report
Author:Lueders, Imke; Ludwig, Carsten; Kasberg, Johanna; Baums, Christoph Georg; Klimke, Kerstin; Dorner, M
Publication:Journal of Avian Medicine and Surgery
Date:Dec 1, 2017
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